Human immunodeficiency virus-1 (HIV-1), the causative agent of AIDS, remains a global health crisis with millions of new infections each year. While modern antiviral therapies can manage HIV-1 disease in many patients, a cure or vaccine remains out of reach. HIV-1 requires a multitude of host factors in order to replicate and the targeting of cellular factors with small molecules or genome engineering has reinvigorated the search for novel HIV-1 therapeutics. Yet despite decades of research, the full armament of host factors hijacked by HIV-1 remain uncharacterized. The use of yeast as a model organism has been pivotal for our current understanding of basic cell biology and disease mechanisms, from human neurodegenerative disorders to human pathogens. I have recently demonstrated that an epistasis mini-array profile (E-MAP), a genetic interaction (GI) analysis technology, in yeast is well suited to uncover host factors of HIV-1 Rev, an essential viral protein. The hypothesis of this proposal is that a quantitative, genome-wide survey of Rev domain-specific cellular GIs will uncover novel HIV-1 host factors and provide key mechanistic insights into their role in Rev-dependent processes. The goal of Aim 1 is to generate a genome-wide E-MAP of Rev in the context of well defined Rev point mutants (a pE-MAP). A genome-wide pE-MAP of Rev mutants that are defective in nuclear location, multimerization, and nuclear export will be generated in S. cerevisiae. With the aid of bioinformatics, these experiments will uncover novel functional interactions between individual Rev domains and cellular genes. Bridging these findings into human CD4+ T cells with functional HIV-1 infectivity assays will further our understanding of how HIV-1 hijacks cellular processes. Aim 2 will establish the role of putative Rev host factors during HIV-1 replication in human cells with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Depleting cells of the expression of putative Rev host factors with CRISPRs will allow the interrogation of their role in HIV-1 infection in various human cell types. Experiments with a Rev-dependent reporter CD4+ T cell line and various Rev functional assays will clarify the mechanism(s) for how the identified host factors affect specific Rev functions. These studies will culminate in determining the role of Rev host factors during HIV-1 infection of primary human CD4+ T cells. Results from this proposal will greatly expand our understanding of HIV-1 host factors and may identify potential anti-HIV-1 therapeutic targets.